In this study, antimicrobial and antibiofilm activities and the chemical composition of Thymus sipyleus BOISS. subsp. sipyleus BOISS. var. davisianus RONNIGER essential oil was evaluated. The essential oil was obtained by hydro-distillation and analyzed by gas chromatography-mass spectrometry. Fourteen compounds were characterized, having as major components thymol (38.31 %) and carvacrol (37.95 %). Minimum inhibitory concentrations (MICs) of oil and the major components were calculated by serial dilution method, and anti-biofilm effects by microplate biofilm assay against five Gram positive (Staphylococcus aureus MU 38, MU 40, MU 46, MU 47, Stahylococcus epidermidis MU 30) and five Gram negative (Pseudomonas aeruginosa MU 187, MU 188, MU 189, Pseudomonas fluorescens MU 180, MU 181) bacteria. It was found that MICs for essential oil, thymol and carvacrol were between 5 and 50 A mu l/ml, 0.125-0.5 A mu g/ml and 0.125-05 A mu l/ml, respectively. The results showed that doses of MIC produced a greater anti-biofilm influence than 0.5, 0.25 and 0.125 MIC. In the presence of essential oil (MIC), the mean biofilm formation value was equal to 67 +/- A 5.5 % for P. aeruginosa MU 188, and essential oil (MIC) inhibition exceeds 60 % for P. aeruginosa biofilms. The results also showed that carvacrol (MIC) was able to induce an inhibition 72.9 +/- A 4.1 % for S.aureus (MU 40) biofilm. In addition, thymol (MIC) showed 68.6 +/- A 5.3 % reduction in biofilm formation of P. fluorescens MU 181. This study demonstrated the antimicrobial and antibiofilm activity of T. sipyleus BOISS. subsp. sipyleus BOISS. var. davisianus RONNIGER essential oil and points out the exceptional efficiency of thymol and carvacrol, which could represent candidates in the treatment of Pseudomonas and Staphylococcus biofilms.